The innate immune system has evolved numerous molecular sensors and signaling pathways to detect, contain and clear viral infections (Takeuchi O and Akira S Immunol Rev 227, 75-86 (2009); Yoneyama M and Fujita T, Rev Med Virol 20, 4-22 (2010); Wilkins C and Gale M Curr Opin Immunol 22, 41-47 (2010); and Brennan K and Bowie A G Curr Opin Microbiol 13, 503-507 (2010); all of which are incorporated by reference herein.) Viruses are sensed by a subset of pattern recognition receptors (PRRs) that recognize evolutionarily conserved structures known as pathogen-associated molecular patterns (PAMPs). Classically, viral nucleic acids are the predominant PAMPs detected by these receptors during infection. These sensing steps contribute to the activation of signaling cascades that culminate in the early production of antiviral effector molecules, cytokines and chemokines responsible for the inhibition of viral replication and the induction of adaptive immune responses (Takeuchi O and Akira S Cell 140, 805-820 (2010), Liu S Y et al, Curr Opin Immunol 23, 57-64 (2011); and Akira S et al, Cell 124, 783-801 (2006); all of which are incorporated by reference herein). In addition to the nucleic acid sensing by a subset of endosome-associated Toll-like receptors (TLR), viral RNA structures within the cytoplasm are recognized by members of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) family, including the three DExD/H box RNA helicases RIG-I, Mda5 and LGP-2 (Kumar H et al, Int Rev Immunol 30, 16-34 (2011); Loo Y M and Gale M, Immunity 34, 680-692 (2011); Belgnaoui S M et al, Curr Opin Immunol 23, 564-572 (2011); Beutler B E, Blood 113, 1399-1407 (2009); Kawai T and Akira S, Immunity 34, 637-650 (2011); all of which are incorporated by reference herein.)
RIG-I is a cytosolic multidomain protein that detects viral RNA through its helicase domain (Jiang F et al, Nature 479, 423-427 (2011) and Yoneyama M and Fujita T, J Biol Chem 282, 15315-15318 (2007); both of which are incorporated by reference herein). In addition to its RNA sensing domain, RIG-I also possesses an effector caspase activation and recruitment domain (CARD) that interacts with the mitochondrial adaptor MAVS, also known as VISA, IPS-1, and Cardif (Kawai T et al, Nat Immunol 6, 981-988 (2005) and Meylan E et al, Nature 437, 1167-1172 (2005), both of which are incorporated by reference herein.) Viral RNA binding alters RIG-I conformation from an auto-inhibitory state to an open conformation exposing the CARD domain, resulting in RIG-I activation which is characterized by ATP hydrolysis and ATP-driven translocation of RNA (Schlee M et al, Immunity 31, 25-34 (2009); Kowlinski E et al, Cell 147, 423-435 (2011); and Myong S et al, Science 323, 1070-1074 (2011); all of which are incorporated by reference herein). Activation of RIG-I also allows ubiquitination and/or binding to polyubiquitin. In recent studies, polyubiquitin binding has been shown to induce the formation of RIG-I tetramers that activate downstream signaling by inducing the formation of prion-like fibrils comprising the MAVS adaptor (Jiang X et al, Immunity 36, 959-973 (2012); incorporated by reference herein). MAVS then triggers the activation of IRF3, IRF7 and NF-κB through the IKK-related serine kinases TBK1 and IKKε (Sharma S et al, Science 300, 1148-1151 (2003); Xu L G et al, Molecular Cell 19, 727-740 (2005); and Seth R B et al, Cell 122, 669-682 (2005); all of which are incorporated by reference herein). This in turn leads to the expression of type I interferons (IFNβ and IFNα), as well as pro-inflammatory cytokines and anti-viral factors (Tamassia N et al, J Immunol 181, 6563-6573 (2008) and Kawai T and Akira S, Ann NY Acad Sci 1143, 1-20 (2008); both of which are incorporated by reference herein.) A secondary response involving the induction of IFN stimulated genes (ISGs) is induced by the binding of IFN to its cognate receptor (IFNα/βR). This triggers the JAK-STAT pathway to amplify the antiviral immune response (Wang B X and Fish E N Trends Immunol 33, 190-197 (2012); Nakhaei P et al, Activation of Interferon Gene Expression Through Toll-like Receptor-dependent and -independent Pathways, in The Interferons, Wiley-VCH Verlag GmbH and Co KGaA, Weinheim F R G (2006); Sadler A J and Wiliams B R, Nat Rev Immunol 8, 559-568 (2008); and Schoggins J W et al, Nature 472, 481-485 (2011); all of which are incorporated by reference herein.)
The nature of the ligand recognized by RIG-I has been the subject of intense study given that PAMPs are the initial triggers of the antiviral immune response. In vitro synthesized RNA carrying an exposed 5′ terminal triphosphate (5′ppp) moiety was identified as a RIG-I agonist (Hornung V et al, Science 314, 994-997 (2006); Pichlmair A et al, Science 314, 997-1001 (2006); and Kim D H et al, Nat Biotechol 22, 321-325 (2004); all of which are incorporated by reference herein). The 5′ppp moiety is added to the end of all viral and eukaryotic RNA molecules generated by RNA polymerization. However, in eukaryotic cells, RNA processing in the nucleus cleaves the 5′ppp end and the RNA is capped prior to release into the cytoplasm. The eukaryotic immune system evolved the ability to distinguish viral ‘non-self’ 5′ppp RNA from cellular ‘self’ RNA through RIG-I (Fujita T, Immunity 31, 4-5 (2009); incorporated by reference herein). Further characterization of RIG-I ligand structure indicated that blunt base pairing at the 5′ end of the RNA and a minimum double strand (ds) length of 20 nucleotides were also important for RIG-I signaling (Schlee M and G Hartmann, Molecular Therapy 18, 1254-1262 (2010); incorporated by reference herein). Further studies indicated that a dsRNA length of less than 300 base pairs led to RIG-I activation but a dsRNA length of more than 2000 bp lacking a 5′ppp (as is the case with poly I:C) failed to activate RIG-I. (Kato H et al, J Exp Med 205, 1601-1610 (2008); incorporated by reference herein).
RNA extracted from virally infected cells, specifically viral RNA genomes or viral replicative intermediates, was also shown to activate RIG-I (Baum A et al, Proc Natl Acad Sci USA 107, 16303-16308 (2010); Rehwinkel J and Sousa C R E, Science 327, 284-286 (2010); and Rehwinkel J et al, Cell 140, 397-408 (2010); all of which are incorporated by reference herein). Interestingly, the highly conserved 5′ and 3′ untranslated regions (UTRs) of negative single strand RNA virus genomes display high base pair complementarity and the panhandle structure theoretically formed by the viral genome meets the requirements for RIG-I recognition. The elucidation of the crystal structure of RIG-I highlighted the molecular interactions between RIG-I and 5′ppp-dsRNA (Cui S et al, Molecular Cell 29, 169-179 (2008); incorporated by reference herein), providing a structural basis for the conformational changes involved in exposing the CARD domain for effective downstream signaling.